Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR.
The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)(6) tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.
Zou
Chao
C
Institute of Organic Chemistry, University of Zurich, Switzerland.
Naider
Fred
F
Zerbe
Oliver
O
D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D003902:Detergents; D008823:Micelles; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; C109491:neuropeptide Y4 receptor